Genes and proteins, and their use

ABSTRACT

According to the present invention, a series of genes are identified in Group B Streptococcus, the products of which may be associated with the outer surface of the organism. The genes, or functional fragments thereof, may be useful in the preparation of therapeutics, e.g. vaccines to immunize a patient against microbial infection.

FIELD OF THE INVENTION

This invention relates to the identification of bacterial genes andproteins, and their use. More particularly, it relates to their use intherapy, for immunisation and in screening for drugs.

BACKGROUND TO THE INVENTION

Group B Streptococcus (GBS), also known as Streptococcus agalactiae, isthe causative agent of various conditions. In particular, GBS causes:

Early Onset Neonatal Infection.

This infection usually begins in utero and causes severe septicaemia andpneumonia in infants, which is lethal if untreated and even withtreatment is associated with a 10-20% mortality rate.

Late Onset Neonatal Infection.

This infection occurs in the period shortly after birth until about 3months of age. It causes a septicaemia, which is complicated bymeningitis in 90% of cases. Other focal infections also occur includingosteomyelitis, septic arthritis, abscesses and endopthalmitis.

Adult Infections.

These appear to be increasingly common and occur most frequently inwomen who have just delivered a baby, the elderly and theimmunocompromised. They are characterised by septicaemia and focalinfections including osteomyelitis, septic arthritis, abscesses andendopthalmitis.

Urinary Tract Infections.

GBS is a cause of urinary tract infections and in pregnancy accounts forabout 10% of all infections.

Veterinary Infections.

GBS causes chronic mastitis in cows. This, in turn, leads to reducedmilk production and is therefore of considerable economic importance.

GBS infections can be treated with antibiotics. However, immunisation ispreferable. It is therefore desirable to develop an immunogen that couldbe used in a therapeutically-effective vaccine.

SUMMARY OF THE INVENTION

The present invention is based on the identification of a series ofgenes in GBS, and also related organisms, the products of which may belocalised on the outer surface of the organism and therefore may be usedas a target for immuno-therapy.

According to one aspect of the invention, a peptide is encoded by anoperon including any of the genes identified herein as pho1-13, pho3-21,pho2-15, pho3-18, pho3-22, pho3-3, pho3-17, pho2-2, pho1-5, pho3-1,pho3-23, pho3-50, pho1-14, pho2-10, pho3-14, pho3-24 and pho3-29,obtainable from Group B Streptococcus, or a homologue or functionalfragment thereof. Such a peptide is suitable for therapeutic use, e.g.when isolated.

The term “functional fragments” is used herein to define a part of thegene or peptide which retains the activity of the whole gene or peptide.For example, a functional fragment of the peptide may be used as anantigenic determinant, useful in a vaccine or in the production ofantibodies.

A gene fragment may be used to encode the active peptide. Alternatively,the gene fragment may have utility in gene therapy, targeting thewild-type gene in vivo to exert a therapeutic effect.

A peptide according to the present invention may comprise any of theamino acid sequences identified herein as SEQ ID NOS. 2, 4, 6, 8, 10,11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33 and 35, or a functionalfragment thereof.

Because of the extracellular or cell surface location, the peptides ofthe present invention may be suitable candidates for the production oftherapeutically-effective vaccines against GBS. The term“therapeutically-effective” is intended to include the prophylacticeffect of vaccines. For example, a vaccine may comprise a peptideaccording to the invention, or the means for its expression, for thetreatment of infection. The vaccine may be administered to females priorto or during pregnancy to protect mother and neonate against infectionby GBS.

According to another aspect of the invention, the peptides or genes maybe used for screening potential antimicrobial drugs or for the detectionof virulence.

A further aspect of this invention is the use of any of the productsidentified herein, for the treatment or prevention of a conditionassociated with infection by a Group B Streptococcal strain.

Although the protein has been described for use in the treatment ofpatients, veterinary uses of the products of the invention are alsoconsidered to be within the scope of the present invention. Inparticular, the peptides or the vaccines may be used in the treatment ofchronic mastitis, especially in cows.

DESCRIPTION OF THE INVENTION

The present invention is described with reference to Group BStreptococcal strain M732. However, all the GBS strains and many otherbacterial strains are likely to include related peptides or proteinshaving amino acid sequence homology with the peptide of M732. Organismslikely to contain the peptides include, but are not limited to, S.pneumoniae, S. pyogenes, S. suis, S. milleri, Group C and Group GStreptococci and Enterococci. Vaccines to each of these may be developedin the same way as described for GBS.

Preferably, the peptides that may be useful for the production ofvaccines have greater than 40% sequence similarity with the peptidesidentified herein. More preferably, the peptides have greater than 60%sequence similarity. Most preferably, the peptides have greater than 80%sequence similarity, e.g. 95% similarity.

Having characterised a gene according to the invention, it is possibleto use the gene sequence to establish homologies in othermicroorganisms. In this way it is possible to determine whether othermicroorganisms have similar outer surface products. Sequence homologiesmay be established by searching in existing databases, e.g. EMBL orGenbank.

Peptides or proteins according to the invention may be purified andisolated by methods known in the art. In particular, having identifiedthe gene sequence, it will be possible to use recombinant techniques toexpress the genes in a suitable host. Active fragments and homologuescan be identified and may be useful in therapy. For example, thepeptides or their active fragments may be used as antigenic determinantsin a vaccine, to elicit an immune response. They may also be used in thepreparation of antibodies, for passive immunisation, or diagnosticapplications. Suitable antibodies include monoclonal antibodies, orfragments thereof, including single chain fv fragments. Methods for thepreparation of antibodies will be apparent to those skilled in the art.

The preparation of vaccines based on attenuated microorganisms is knownto those skilled in the art. Vaccine compositions can be formulated withsuitable carriers or adjuvants, e.g. alum, as necessary or desired, andused in therapy, to provide effective immunisation against Group BStreptococci or other related microorganisms. The preparation of vaccineformulations will be apparent to the skilled person.

More generally, and as is well known to those skilled in the art, asuitable amount of an active component of the invention can be selected,for therapeutic use, as can suitable carriers or excipients, and routesof administration. These factors will be chosen or determined accordingto known criteria such as the nature/severity of the condition to betreated, the type or health of the subject etc.

The products of the present invention were identified as follows:

A partial gene library of GBS (strain M732) chromosomal DNA was preparedusing the plasmid vectors pFW-phoA1, pFW-phoA2 and pFW-phoA3(Podbielski, A. et al. 1996. Gene 177:137-147). These plasmids possess aconstitutive spectinomycin adenyltransferase antibiotic resistancemarker, which confers a high level of spectinomycin resistance and istherefore easily selected. Furthermore, these vectors contain atruncated (leaderless) Escherichia coli phoA gene for alkalinephosphatase. The three vectors differ only with respect to the readingframe in which the leaderless phoA gene exists, as compared to anupstream in-frame BamHI restriction enzyme site. Because this truncatedE. coli phoA gene lacks the appropriate leader sequence for export ofthis enzyme across the bacterial membrane, extracellular alkalinephosphatase activity is absent when these plasmids are propagated in anE. coli phoA mutant (e.g. strain DH5α). The chromogenic alkalinephosphatase substrate, XP (5-bromo-4-chloro-3-indolyl-phosphate), doesnot enter intact bacterial cells and therefore only exported or surfaceassociated alkaline phosphatase activity can be detected. When exportedor surface associated alkaline phosphatase activity is present, thechromogenic XP substrate is cleaved to yield a blue pigment and thecorresponding bacterial colonies can be identified by their blue colour.

Plasmid DNA was digested to completion with BamHI and dephosphorylatedusing shrimp alkaline phosphatase. GBS genomic DNA was partiallydigested with Sau3AI, size fractionated on a sucrose gradient andfragments <1 kb in size were ligated into the prepared pFW-phoA vectors.E. coli strain DH5α was chosen as the cloning host since it lacks afunctional phoA gene. Recombinant plasmids were selected on Luria agarcontaining 100 μg/ml of spectinomycin and 40 μg/ml of the chromogenic XPsubstrate. E. coli transformants harbouring plasmids containing GBSinsert DNA that complements the export signal sequence of the leaderlessphoA gene were identified by the blue colour of the colonies.Approximately 30000 different recombinant plasmids containing GBS insertDNA were screened in this manner and 83 recombinant plasmids, whichcomplemented the leaderless phoA, were chosen for further study.

From these experiments, several clones were selected each containing aplasmid containing a gene (or part thereof), which complemented theleaderless phoA.

Having identified the gene in each clone it is then possible to obtainthe full-length gene sequence, as follows.

Using the identified and sequenced gene fragment, oligonucleotideprimers were designed for genomic DNA sequencing. These primers weredesigned so as to sequence in an ‘outward’ direction from the obtainedsequence. Once read, the sequence obtained was checked to see if the 5′and 3′ termini of the gene had been reached. The presence of thesefeatures was identified by checking against homologous sequences, andfor the 5′ end the presence of an AUG start codon (or acceptedequivalent) preceded by a Shine-Dalgarno consensus sequence, and for the3′ end, the presence of a translation termination (Stop) codon.

Upon identification of the full-length gene, primers were designed foramplification of full-length product. Primers used included restrictionenzyme recognition sites (NcoI at the 5′ end and EcoO109I at the 3′ end)to allow subsequent cloning of the product into the Lactococcalexpression system used.

PCR was carried out using the primers, and the products cloned into apCR 2.1 cloning vector (In Vitrogen). Following confirmation of thepresence of the cloned fragment, the DNA was excised using therestriction enzymes NcoI and EcoO109I.

The vector into which this fragment was inserted was a modified versionof pNZ8048 (Kuipers, O. P. et al. (1998) J. Biotech 64: 15-21). Thisvector, harbouring a lactococcal origin of replication, achloramphenicol resistance marker, an inducible nisin promoter and amulticloning site was altered by the replacement of the multicloningsite with two 10× His tags, flanked on the 5-most end with an NcoI site,split in the middle with a multicloning site (including an EcoO109Isite), and a Stop (termination) codon at the 3′ end of the His tags.

The gene of interest was inserted so that a 10× His tag was in the 3′position relative to the coding region. Following transformation of therecombinant plasmid into L. lactis (strain NZ9000-Kuipers, O. P. et al.(1998) supra), a 400 ml liquid culture was set up and translation of theprotein was induced by the addition of nisin to the culture. After a 2hour incubation, the cells were harvested and lysed by bead beating. Theresultant lysate was cleared by centrifugation, then passed over a metalaffinity (Talon, Clonetech) column. The column was washed repeatedlybefore bound proteins were eluted with Imidazole.

To identify fractions containing the His-tagged recombinant protein, analiquot from each fraction was analysed by SDS-PAGE, Western blotted andprobed with anti-His antibodies.

The recombinant protein obtained was then used to immunize New Zealandwhite rabbits, with pre-immune sera being harvested prior toimmunisation. Following a boost, the rabbits were sacrificed and seracollected. This sera was used in Western blots, ELISA and animalprotection models.

Using the sera obtained from the animal studies, immunosorption studieswere carried out.

Group B Streptococcus was grown in 20 ml Todd Hewitt broth (THB) for 8hours, harvested and resuspended in 5 ml PBS. 50 μl aliquots of thiswere used to coat wells in a 96 well plate (Nunc Immuno-Sorb). This wasleft at 4° C. overnight to allow for absorbance of the bacteria onto theplate. Plates were washed twice with PBS, then blocked with 3% BSA inPBS for 1 hr at 37° C. Plates were again washed. Serial 10 folddilutions of the sera were made in PBS and 50 μl of these dilutions wereadded to the wells of the plate, in duplicate. The plate was covered andincubated for 1 hr at 37° C. The plate was washed, then 50 μlanti-rabbit alkaline phosphatase conjugated secondary antibody at aconcentration of 1:5000 was added to each well. Following incubation at37° C. for an hour, the plate was washed again. 50 μl substrate (PNPP)was added to each well, and the reaction allowed to proceed for 30 minbefore the absorbance was read at 405 nm.

Animal protection studies were also carried out to test theeffectiveness of protection on the immunised rabbits.

GBS M732 was grown up in THB until mid-log phase wasreached—approximately 5 hours. Cells were counted in a counting chamber,and bacteria were diluted to give a concentration of 2×10⁷ bacteria perml in pre-immune or test sera. 50 μl of this was injected via theintraperitoneal route into 0-1 day old mice. The mice were observed forsurvival over 48 hours.

The following Examples illustrate the invention.

EXAMPLE 1

A first clone contained a gene sequence identified herein as SEQ ID NO.1, with an amino acid sequence identified as SEQ ID NO. 2, andclassified as pho1-13.

A comparison of the amino acid sequence of pho1-13 was performed.

Homologues to the GBS pho1-13 gene product can be identified inStreptococcus pyogenes, S. pneumoniae, S. salivarius, Escherichia coli,Yersinia enterocolitica, Aquifex aeolicus, Helicobacter pylori andHaemophilus influenzae. The S. pyogenes and S. pneumoniae homologueswere identified from genome sequence data and no annotations wereavailable as to the identity of the gene or gene products. In all othercases, the above homologues can be identified as ATP-dependent Clpprotease proteolytic subunits. The catalytic activity of Clp proteasesresults in the hydrolysis of proteins to small peptides in the presenceof ATP and magnesium (Giffard, P. M. et al. 1993. J. Gen. Microbiol.139:913-920). Furthermore, the ClpP component of Clp proteases has beenshown to be induced as part of the heat shock response (Kroh, H. E. andL. D. Simon. 1990. J. Bacteriol. 172:6026-6034) and it is probable thatthis subunit or the complete proteolytic domain would associated withthe bacterial surface.

Immunisation studies, carried out as described above, yielded thefollowing results.

No animals surviving at time (hrs) Treatment No animals 24 48 PBS 10 7 0Pre-immunised 37 13 0 Immunised 38 17 9

EXAMPLE 2

A second clone was selected containing a plasmid designated pho1-14.This plasmid contained a gene (or part thereof), which complemented theleaderless phoA. The nucleotide and deduced amino acid sequences areshown as SEQ ID NOS. 3 and 4, respectively.

A comparison of the amino acid sequence of pho1-14 was performed.

Homologues to the GBS pho1-14 gene product can be identified inStreptococcus pyogenes, Enterococcus faecalis and Streptococcuspneumoniae. These homologues were identified from genome sequence dataand no annotations were available as to the identity of the gene or geneproducts. Additionally, two possible homologues were also identifiedfrom Shigella flexneri (SpaR) and Yersinia pseudotuberculosis (YscT).These latter two homologues are related proteins, believed to beanchored in the bacterial membrane (Bergman, T. et al. 1994. J.Bacteriol. 176:2619-2626). In S. flexneri, the product of the spaR genehas been shown to be important for invasion of epithelial cells(Sasakawa, C. et al. 1993. J. Bacteriol. 175:2334-2346). Furthermore,the product of the spaR gene is also required for surface presentationof invasion plasmid antigens. The analogous protein in Y.pseudotuberculosis is a component of the Yop secretion system and isalso important for virulence in this organism.

EXAMPLE 3

A third clone was selected containing a plasmid designated pho1-5. Thisplasmid contained a gene (or part thereof), which complemented theleaderless phoA. The nucleotide and deduced amino acid sequences areshown as SEQ ID NOS. 5 and 6.

A comparison of the amino acid sequence of pho1-5 was performed.

Homologues to the GBS pho1-5 gene product can only be identified instreptococcus pyogenes and Staphylococcus carnosus (sceA). The S.pyogenes homologue was identified from genome sequence data and noannotations were available as to the identity of the gene or geneproducts. Furthermore, little information is available on the functionof the sceA gene product from S. carnosus. The sceA gene product showssome sequence similarity to the aggregation promoting protein fromLactobacillus gasseri. Based on analysis of the sceA gene product, thismolecule contains a well-conserved signal sequence and is apparentlysecreted or associated with the bacterial cell surface.

EXAMPLE 4

A further clone was selected containing a plasmid designated pho3-3.This plasmid contained a gene (or part thereof), which complemented theleaderless phoA. The nucleotide and deduced amino acid sequences areshown as SEQ ID NOS. 7 and 8.

A comparison of the amino acid sequence of pho3-3 was performed.

Homologues to the GBS pho3-3 gene product can be identified inStreptococcus mutans (rmiC), (cpsM) S. pneumoniae and S. pyogenes. TheS. pyogenes homologue was identified from genome sequence data and noannotations were available as to the identity of the gene or geneproduct. In S. pneumoniae, the homologue can be identified asdTDP-4-keto-6-deoxy glucose-3,5-epimerase. In the other two cases, theabove homologues can be identified as dTDP-4-keto-L-rhamnose reductase(rmlC). In S. mutants, the gene encoding this enzyme, rmlC, is part ofthe rml locus. The rml locus consists of three genes which exhibitsignificant similarity to enzymes involved in the biosynthesis ofdTDP-rhamnose, the immediate precursor of the rhamnose component in theS. mutans polysaccharide capsule (Tsukioka, Y. et al. 1997. J.Bacteriol. 179:1126-1134). An analogous locus has also been identifiedin S. pneumoniae (Coffey, T. J. et al. 1998. Mol. Micobiol. 17:73-83).Almost all Streptococci characteristically possess rhamnose in theircell wall associated polysaccharides (Schleifer, K. H. and R.Kilper-Bälz. 1987. Syst. Appl. Microbiol. 10:1-19), and it is highlyprobable that dTDP-4-keto-L-rhamnose reductase would be associated withthe outer surface in Streptococci.

EXAMPLE 5

A further clone was selected containing a plasmid designated pho2-10.This plasmid contained a gene (or part thereof), which complemented theleaderless phoA.

The nucleotide sequence is shown as SEQ ID NO. 9. From this, upstreamand downstream coding regions were identified, and the deduced aminoacid sequences shown as SEQ ID NOS. 10 and 11.

A comparison of the amino acid sequences of pho2-10 was performed.

Homologues to the GBS pho2-10 gene product can be identified inStreptococcus pyogenes, Enterococcus faecalis, Debaryomyces occidentalis(hatI) and Escherichia coli (trkD). The S. pyogenes and E. faecalishomologues were identified from genome sequence data and no annotationswere available as to the identity of the gene or gene products. In theyeast D. occidentalis, the hak1 gene is a homologue of the trkD genefrom E. coli (Banuelos, M. A. et al. 1995. EMBO J. 14:3021-3027). Thetrkd gene of E. coli is part of the kup potassium uptake system. Thespecific homolog identified here is the kup system potassium uptakeprotein. The kup system is a constitutive potassium uptake system in E.coli. The kup system potassium uptake protein contains a highlyhydrophobic N-terminus that is predicted to span the membrane at least12 times. Kup is not homologous to other known membrane proteinsequences. There is no indication of ATP binding, and it is proposedthat the system is driven by a chemiosmotic gradient (Schleyer, M. & E.P. Bakker, 1993. J. Bacteriol. 175:6925-6931).

EXAMPLE 6

A further clone was selected containing a plasmid designated pho2-15.This plasmid contained a gene (or part thereof), which complemented theleaderless phoA. The nucleotide and deduced amino acid sequences of thegene are shown as SEQ ID NOS. 12 and 13.

A comparison of the amino acid sequence of pho2-15 was performed.

Homologues to the GBS pho2-15 gene product can be identified inStreptococcus pyogenes, Streptococcus pneumoniae, Enterococcus faecalisand Escherichia coli (gatC and SgcC). The S. pyogenes, S. pneumoniae andE. faecalis homologues were identified from genome sequence data and noannotations were available as to the identity of the gene or geneproducts. In E. coli, the gatC and sgcC gene products can be identifiedas being the IIC component of phosphoenolypyruvate-dependent sugarphosphotransferase systems (PTS), a major carbohydrate active-transportsystem. In PTS systems, the IIC component is typically involved inbinding of extracellular carbohydrates and forms a complex with the IIDcomponent to constitute a membrane channel (Nobelmann, B. and J. W.Lengeler. 1995. Biochim. Biophys. Acta 1262:69-72).

EXAMPLE 7

A further clone was selected containing a plasmid designated pho2-2.This plasmid contained a gene (or part thereof), which complemented theleaderless phoA. The nucleotide and deduced amino acid sequences of thegene are shown as SEQ ID NOS. 14 and 15, respectively.

A comparison of the amino acid sequence of pho2-2 was performed.

Homologues to the GBS pho2-2 gene product can be identified inEnterococcus faecalis, Escherichia coli (malK and afuC), Bacillussubtilis (glnO), Haemophilus influenzae (yebM and potA), Streptococcuspyogenes, Streptococcus pneumoniae and Salmonella typhimurium (malK).The E. faecalis, S. pyogenes and S. pneumoniae homologues wereidentified from genome sequence data and no annotations were availableas to the identity of the gene or gene products. In all other cases,homologues represented ATP-binding transport proteins that are part ofABC type transporters. Many of the components of ABC type transportersare membrane or cell surface associated, as these systems are involvedin the transport of macromolecules from the extracellular environment tothe intracellular compartment.

EXAMPLE 8

A further clone was selected containing a plasmid designated pho3-14.This plasmid contained a gene (or part thereof), which complemented theleaderless phoA. The nucleotide and deduced amino acid sequences of thegene are shown as SEQ ID NOS. 16 and 17.

A comparison of the amino acid sequence of pho3-14 was performed and nohomologues could be identified in any of the public databases. Onehomologue to the GBS pho3-14 gene product can be identified inStreptococcus pyogenes, but this homologue was identified from genomesequence data and no annotations were available as to the identity ofthe gene or gene product. Using this S. pyogenes homologue to search thepublic databases yielded no further information. Since the pho3-14product complemented the leaderless phoA gene, it can be concluded thatthis protein (or part thereof) would most probably be locatedextracellularly.

EXAMPLE 9

A further clone was selected containing a plasmid designated pho3-17.This plasmid contained a gene (or part thereof), which complemented theleaderless phoA. The nucleotide and deduced amino acid sequences of thegene are shown as SEQ ID NOS. 18 and 19.

A comparison of the amino acid sequence of pho3-17 was performed.

Homologues to the GBS Pho3-17 gene product can be identified inStreptococcus mutans and Lactococcus lactis, with similarity being shownto N-acetyl muramidase. Similarity is also seen with an unidentifiedgene, yubE from Bacillus subtilis.

N-acetylmuramidase is an autolysin that is involved in cell division.Using this limited information along with the fact that pho3-17complemented the leaderless phoA gene, it can be concluded that thepho3-17 product would most probably be located extracellularly.

EXAMPLE 10

A further clone was selected containing a plasmid designated pho3-18.This plasmid contained a gene (or part thereof), which complemented theleaderless phoA. The nucleotide and deduced amino acid sequences of thegene are shown as SEQ ID NOS. 20 and 21.

A comparison of the amino acid sequence of pho3-18 was performed.

Homologues to the GBS pho3-18 gene product can be identified inStreptococcus pyogenes and Streptococcus pneumoniae. These homologueswere identified from genome sequence data and no annotations wereavailable as to the identity of the gene or gene products. Using theseS. pyogenes and S. pneumoniae homologues to search the public databasesshowed some similarity to outer surface and membrane spanning proteins.Since the ORF3-18 product complemented the leaderless phoA gene, it canbe concluded that this protein (or part thereof) would most probably belocated extracellularly.

EXAMPLE 11

A further clone was selected containing a plasmid designated pho3-1.This plasmid contained a gene (or part thereof), which complemented theleaderless phoA. The nucleotide and deduced amino acid sequences of thegene are shown as SEQ ID NOS. 22 and 23.

A comparison of the amino acid sequence of pho3-1 was performed.

Homologues to the GBS pho3-1 gene product can be identified inStreptococcus pyogenes, streptococcus pneumoniae, Bacillus subtilis(yutD) and Enterococcus faecalis. The S. pyogenes, S. pneumoniae and E.faecalis homologues were identified from genome sequence data and noannotations were available as to the identity of the gene or geneproducts. In B. subtilis, the function of the yutD gene product isunknown. It can be noted however, that the yutD gene is located on theB. subtilis chromosome in a region containing genes involved in cellwall synthesis. The fact that this DNA sequence complemented theleaderless phoA gene suggests that this gene product is extracellularlylocated.

EXAMPLE 12

A further clone was selected containing a plasmid designated pho3-21.This plasmid contained a gene (or part thereof), which complemented theleaderless phoA. The nucleotide and deduced amino acid sequences of thegene are shown as SEQ ID NOS. 24 and 25.

A comparison of the amino acid sequence of pho3-21 was performed.

Homologues to the GBS pho3-21 gene product can be identified inStreptococcus pyogenes, Streptococcus pneumoniae, Lactobacillusfermentum (bspA) and Lactobacillus reuteri (cnb). The S. pyogenes and S.pneumoniae homologues were identified from genome sequence data and noannotations were available as to the identity of the gene or geneproducts. In L. fermentum, the bspA gene product has been identified asbeing a basic cell surface-located protein that has some sequencesimilarity to family III of the bacterial solute-binding proteins(Turner, M. S. et al. 1997. J. Bacteriol. 179:3310-3316). In L. reuteri,the cnb gene product has been identified as a collagen binding proteinthat has some sequence similarity to the solute-binding component ofbacterial ABC transporters (Roos, S. et al. 1996. FEMS Microbiol. Lett.144:33-38).

EXAMPLE 13

A further clone was selected containing a plasmid designated pho3-22.This plasmid contained a gene (or part thereof), which complemented theleaderless phoA. The nucleotide and deduced amino acid sequences of thegene are shown as SEQ ID NOS. 26 and 27.

A comparison of the amino acid sequence of pho3-22 was performed.

Homologues to the GBS pho3-22 gene product can be identified inEnterococcus faecalis, Streptococcus equisimilis (lppC), Pseudomonasfluorescens (oprI) and Streptococcus thermophilus (orf142). The E.faecalis homolog was identified from genome sequence data and noannotations were available as to the identity of the gene or geneproducts. In S. equisimilis, the lppC gene product has been identifiedas being a lipoprotein that is homologous to the E(P4) outer membraneprotein from Haemophilus influenzae (Gase, K. et al. 1997. Med.Microbiol. Immunol. 186:63-73). Likewise, the P. fluorescens oprI geneencodes a major outer membrane lipoprotein (Cornelis, P. et al. 1989.Mol. Microbiol. 3:421-428). In S. thermophilus, the orf142 product hasbeen putatively identified as a cell surface exposed lipoprotein thatmay act as a receptor for the bacteriophages TP-J34 and Sfi21 (Neve, H.et al. 1998. Virology 241:61-72). The ORF3-22 product showed goodsimilarity to the above homologues, particularly at the N-terminus. Thisis most likely the region required for complementation of the leaderlessphoA gene, and therefore serves as a leader sequence.

EXAMPLE 14

A further clone was selected containing a plasmid designated pho3-23.This plasmid contained a gene (or part thereof), which complemented theleaderless phoA. The nucleotide and deduced amino acid sequences of thegenes are shown as SEQ ID NOS. 28 and 29.

A comparison of the amino acid sequence of pho3-23 was performed.

Homologues to the GBS pho3-23 gene product can be identified inStreptococcus pyogenes, Streptococcus pneumoniae, Enterococcus faecalisand Streptococcus mutans (perM). The S. pyogenes, S. pneumoniae and E.faecalis homologues were identified from genome sequence data and noannotations were available as to the identity of the gene or geneproducts. In S. mutans, the perM gene product has been presumptivelyidentified as a permease, but no other information is available as tothe function of this protein. Considering that the pho3-23 coding regioncomplements the leaderless phoA gene, it can be concluded that thepho3-17 gene product would most probably be located extracellularly.

EXAMPLE 15

A further clone was selected containing a plasmid designated pho3-24.This plasmid contained a gene (or part thereof), which complemented theleaderless phoA. The nucleotide and deduced amino acid sequences of thegene are shown as SEQ ID NOS. 30 and 31.

A comparison of the amino acid sequence of pho3-24 was performed.

Homologues to the GBS pho3-24 gene product can be identified inStreptococcus mutans (dltB), Streptococcus pneumoniae, Streptococcuspyogenes, Enterococcus faecalis, Lactobacillus casei (dltB) and Bacillussubtilis (dltB). The S. pneumoniae, S. pyogenes and E. faecalishomologues were identified from genome sequence data and no annotationswere available as to the identity of the gene or gene products. In S.mutans, L. casei and B. subtilis, the dltB gene product has beenidentified as being a basic membrane protein that is involved in thetransport of activated D-alanine through the cell membrane. The dltBgene product is involved in the biosynthesis of D-alanyl-lipoteichoicacid (Heaton, M. P. and F. C. Neuhaus. 1992. J. Bacteriol.174:4707-4717). In L. casei and B. subtilis, the dltB gene product isbelieved to contain at least 9 membrane spanning domains, indicatingthat the protein or portions thereof are exposed to the outside of thecell.

EXAMPLE 16

A further clone was selected containing a plasmid designated pho3-29.This plasmid contained a gene (or part thereof), which complemented theleaderless phoA. The nucleotide and deduced amino acid sequences of thegene are shown as SEQ ID NOS. 32 and 33.

A comparison of the amino acid sequence of pho3-29 was performed.

Homologues to the GBS pho3-29 gene product can be identified in Borreliaburgdorferi (p23 or ospC), Bacillus brevis (owp) and Pseudomonasaeruginosa (oprI). Although these homologues are not related to eachother, they all represent major outer surface proteins. In B.burgdorferi, the ospC gene product has been identified as being a 23-kDaprotein that is the immunodominant antigen on the surface of thisbacterium (Padula, S. J. et al. 1993. Infect. Immun. 61:5097-5105). Theowp gene product from B. brevis is one of two major cell wall proteinsinvolved in the surface layer lattice (Tsuboi, A. 1988. J. Bacteriol.170:935-945). Finally, the oprI gene from P. aeruginosa encodes a majorouter membrane lipoprotein precursor (Saint-Onge, A. et al. 1992. J.Gen. Microbiol. 138:733-741).

EXAMPLE 17

A further clone was selected containing a plasmid designated pho3-50.This plasmid contained a gene (or part thereof), which complemented theleaderless phoA. The nucleotide and deduced amino acid sequences of thegene are shown as SEQ ID NOS. 34 and 35.

A comparison of the amino acid sequence of pho3-50 was performed.

Homologues to the GBS pho3-50 gene product can be identified in avariety of Streptococci (penA, pbp2B, pbpB2), Borrelia burgdorferi(pbp2), Enterococcus faecalis(pbpC), Staphylococcus aureus (pbpA),Mycobacterium laprae(pbpB) and Helicobacter pylori (pbp2). In all cases,the above homologues can be identified as penicillin binding proteins(PBPs). Genes encoding penicillin binding proteins are often located ina cluster of genes associated with cell wall synthesis (Pucci, M. J. etal. 1997. J. Bacteriol. 179:5632-5635). Furthermore, PBPs are typicallyintegrated into the cell wall of a bacterium with some or all of theprotein being exposed on the outer bacterial surface.

1-19. (canceled)
 20. An isolated polypeptide comprising an amino acid sequence which has greater than 95% similarity to SEQ ID NO:
 23. 21. The polypeptide of claim 20, wherein said amino acid sequence comprises SEQ ID NO:
 23. 22. The polypeptide of claim 21, wherein said amino acid sequence is encoded by SEQ ID NO: 22
 23. The polypeptide of claim 20, further comprising a heterologous polypeptide.
 24. A composition comprising the polypeptide of claim 20 and a carrier.
 25. The composition of claim 20, further comprising an adjuvant.
 26. The composition of claim 25, wherein said adjuvant is alum.
 27. The composition of claim 20, which induces an immune response against said polypeptide when administered in an animal.
 28. The composition of claim 27, wherein said immune response is a humoral immune response.
 29. The composition of claim 27, wherein said immune response is a cell mediated immune response. 